Ligand-induced trafficking of G protein-coupled receptors (GPCRs) and their interaction with associated proteins are key steps regulating receptor-mediated signal transduction and cellular responsiveness, u opioid receptor ([unreadable]OR) mediates the effects of opiates, potent drugs used for pain control, including analgesia, opioid bowel syndrome (constipation and abdominal pain), and tolerance. We showed that a) [unreadable]OR endocytoses in enteric neurons in response to endogenously released opioids and to opiates, except morphine, b) chronic [unreadable]OR activation confers morphine the ability to trigger [unreadable]OR endocytosis and induces over expression and translocation of dynamin, an intracellular protein that is important for receptor internalization, c) the magnitude of [unreadable]OR endocytosis induced by high efficacy opiates does not change in a condition of opioid tolerance, d) N-Methyl-D-Aspartate receptors (NMDA-Rs) are likely to be involved in the [unreadable]OR-mediated impairment of gastrointestinal transit induced by prolonged [unreadable]OR activation. This application will test the hypotheses that: 1) prolonged exposure to [unreadable]OR ligands induces changes in the intracellular machinery regulating [unreadable]OR trafficking in enteric neurons and cellular signaling located downstream of the receptor, and 2) NMDA-R activation mediates the development of ileus/constipation induced by chronic [unreadable]OR activation. Specific Aim 1 will establish which intracellular proteins regulate [unreadable]OR trafficking following acute and chronic activation. Specific Aim 2 will test whether agonist-activation of [unreadable]OR stimulates MAP kinase cascade in enteric neurons through a mechanism requiring receptor internalization and whether sustained [unreadable]OR endocytosis following chronic [unreadable]OR activation is associated with persistent MAP kinase activity. Specific Aim 3 will test the role of NMDA-Rs in mediating the effects of chronic opiate treatment in gastrointestinal motility and at the receptor level. Relevance: A better knowledge of how enteric neurons respond and react to chronic opiate drugs will advance our understanding of opioid bowel syndrome, a condition affecting patients receiving chronic opiates for pain, and may facilitate the development of novel therapeutic approaches that reduce the side effects but preserving the efficacy of these drugs.